Differential persistence of neutralizing antibody against SARS-CoV-2 in post immunized Bangladeshi population

Development of effective vaccines have been immensely welcomed by the world to prevent the transmission of SARS-CoV-2. However, the duration and clinical implications of antibody-mediated natural immunity in SARS-CoV-2 have not been adequately elucidated alongside some other immune system transforming factors. In a cohort study, we measured NAb titer following the 2nd immunization dosage of the CoviShield (AZD1222) vaccine. The enzyme-linked immunoassay was used to look for SARS-CoV-2—specific NAb. We measured NAb at 30 days after the 2nd dosage of immunization and > 96% titer was detected in 42.9% of subjects, but only 5.1% of subjects retained the same level after 180 days. The median NAb titer dropped significantly, from 92% at 30 days to 58% at 180 days (p < 0.001). Besides, there were significant differences observed in NAb titer after 180 days by age, sex, COVID-19 infection, tobacco use, and asthma patients. However, SARS-CoV-2 infection along with two dosages of immunization upheld NAb titer (p < 0.001) even at the end of the study period.

Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2) has continued to show a devastating impact on the global population particularly on the immunosuppressed and people with co-morbidity [1][2][3] . This catastrophic circumstance has been prevalent until effective immunization were introduced. In this process, virusspecific neutralizing antibody (NAb) is a very important factor for reducing viral replication and increasing viral clearance 4,5 . Neutralizing antibodies primarily target the SARS-CoV-2 S protein's receptor-binding domain (RBD) [6][7][8] , thereby inhibiting viral entrance into the cell. In the case of SARS CoV-2 infection 9 or immunization 10 , neutralizing antibodies emerge quickly in the human circulatory system and lasts for few months 11,12 . Though this doesn't promise ultimate protection as several variants of concern (VOCs) have evolved by mutating the S gene of the virus.
The CoviShield COVID-19 (AZD1222) (C19VAZ) vaccine, formerly known as ChAdOx1 nCoV-19, is made from a weakened version of a common cold virus (ChAdOx1), where the genetic material has been added to produce SARS-CoV-2 Spike glycoprotein 13 . According to the World Health Organization (WHO), the AZD1222 vaccine has been shown to be 63.09% effective against symptomatic SARS-CoV-2 infection, which is correlated to longer dosage intervals within the 8-12 weeks range for better vaccine efficacy. The Bangladesh government has initiated mass-level vaccination since early February 2021 using AZD1222. Though the mass level of vaccination is ongoing, the transmission of the disease is still in an oscillating phase. Nonetheless, protective immunity after immunization is currently unclear as the immune responses are fading away and new viral variants are spreading at the same time 14 . This raises the dilemma of designing a vaccine that has induced selection pressure for the formation of a viral variation as well as immune response convalescence 15 . Although memory B cells and T cells both play a part in the fight against SARS-CoV-2, there is good evidence that NAb play a major role since passive immunization can lower the severity of the disease 16 . As a result, we have concentrated our research on Sample collection. All subjects had venous blood samples taken in the amount of 5 ml in Vacutainer tubes after completing the consent form. The serum was collected and tested after spinning the blood at 4000 rpm for 10 min at room temperature. The serum samples were then aliquoted and stored at − 80 °C until the tests were performed.
Ethical approval. Approval of this study protocol and ethical clearance was obtained from the Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources, under supervision of Institute of Biological Sciences (IBSc), University of Rajshahi, Rajshahi, Bangladesh (Memo number: 58/320/IAMEBBC/IBSc). Further this was followed in accordance with guidelines of IAMEBBC and as per the protocol all the participants provided written informed consent. SARS-CoV-2 specific neutralizing antibody detection in plasma/serum. The COVID-19 Neutralizing Antibody Microlisa test kits supplied by J. Mitra & Co. Pvt. Ltd., New Delhi, India, were used to test SARS-CoV-2 specific NAb in serum samples according to the manufacturer's recommendations (Lot No. ECN020521). The assay kit was targeted to detect neutralizing antibodies generated against SARS-CoV-2 in human serum/ plasma in an in vitro semi-quantitative manner, preventing the interaction between the viral Spike glycoprotein's receptor binding domain (RBD) and the cell surface receptor angiotensin converting enzyme-2 (ACE2). The detection protocols and routines adhered to the manufacturer's instructions (https:// jmitra. co. in/ wp-conte nt/ uploa ds/ 2021/ 10/ Instr uction-Manual-Covid-19-Neutr alizi ng-Antib odies-Micro lisa. pdf) are as follows: To begin, 20 μl of sample/control and 180 μl of sample diluent buffer (included in the kit) was taken in a clean 1.5 ml micro-centrifuge tube (diluted serum sample, negative control and positive control at a volume ratio of

Results
Trial population. Information on the 531 subjects such as demographic data is shown in Table 1, the subjects had a median age of 42 years (IQR, [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50], with 42% (222) being female and 27% (141) being 50 years old and having received the 2nd dosage of immunization. More than half of the subjects (56%) were medical professionals. Among the total subjects, 18.6% used tobacco, and 8.5% were on mild steroid therapy due to various clinical complications. Furthermore, diabetes, hypertension, and asthma were reported by around 12%, 25%, and 9% of the subjects, respectively (Table 1). Antiviral humoral immunity, which is infrequently apparent more than a year after hospitalization, wanes over time, according to studies of MERS and SARS-CoV beta corona virus infections, two viruses closely related to SARS-CoV-2 17,18 . It is unknown whether SARS-CoV-2 NAb decline at the same pace as SARS-CoV NAb. However, the durability of protective immunity is unknown at this time, primary immune responses are inevitably waning 11,14,19 and there is continual propagation of increasingly dangerous virus variations that may elude both vaccine-induced and convalescent immune responses 19 . Only a few studies have looked into the path of neutralization titers after two dosage of immunization 20 .
NAb levels were first assessed immediately after the second immunization dosage. After 30 days, 42.9% of subjects had NAb levels greater than 96%, but only 5.1% had the same level after 180 days. At the same time, for the vast majority of people, the level had dropped significantly to 70% (See Fig. 1 We discovered that individuals in the older age groups had a faster rate of NAb titer decline than those in the younger age groups. Similarly, 180 days after immunization, the NAb titer in the same group decreased (Fig. 2), which was alarming. NAb titer decreased at a higher rate in the older age groups than in the younger age groups (p = 0.001).
Gender discrepancy in COVID-19 disease severity shows higher mortality rate in male than female 21,22 , though Investigated Gender's impact on immunological memory shows that males have higher Spikes in IgG, nucleocapsid, and RBD IgG than females 11,23 . However, we discovered that the female had a slightly lower response to NAb production after immunization although not significant (p = 0.088) (Fig. 3). We presume that this is an outcome of technical error during vaccination because the female Bangladeshi wore tight clothing where access to the deltoid muscle was harder for vaccine providers. Personal communication with the vaccine providers also confirmed this observation. This is why some females had fewer NAb than after 30 days, though the waning ratio of NAb in females remained similar to males after 180 days. www.nature.com/scientificreports/ We further divided our study group on the basis of tobacco use (in the form of smoking, we only considered male in our country's context) and individuals on corticosteroids in inhaler form for complications like asthma and COPD. When compared to their non-smoking peers, vaccinated people who smoked or used corticosteroids have a lower NAb titer after immunization (Figs. 4 and 5). Furthermore, their NAb titer had fallen significantly (p < 0.001) after 180 days of immunization. The effects of corticosteroids were expected as they have an immune suppressant effect. We assume that the smoker group may have similar immune suppression due to their longtime smoking habit 24 .   www.nature.com/scientificreports/ In a mass immunization initiative, both front-line and academic staff were targeted for vaccination at the same time. This immunization program was in effect when the country was under lockdown and SARS-CoV-2 transmission was at its peak. In our study group, some of the medical professionals were survivors of the SARS-CoV-2 infection. However, medical professionals working in the hospital during the lockdown were more likely to be exposed to the virus than the university faculty members who were staying at home. Though the medical professionals were vaccinated twice, some of the doctors and nurses reported mild or subclinical infection of SARS-CoV-2 after immunization. Whereas, a very few University Faculty member reported the infection either pre or post immunization. Once we compared NAb titter between these two group medical professionals has a greater Nab responses than the university faculty group among the sampled subjects (Fig. 6). This is potentially because of the natural infection with SARS-CoV-2.
We further divided our subjects on the basis of type 2 Diabetes and BMI. Considering the type 2 Diabetes, the immunized people with type 2 Diabetes, shows a lower NAb titer after immunization (Fig. 7). Among subjects having type-2 Diabetes, NAb titers significantly (p < 0.001) decreased with a higher rate than subjects without Type-2 Diabetes. Again when considering the BMI, those who have BMI ≥ 30, have lower NAb titer after immunization (Fig. 8). We observed that the subjects having BMI ≥ 30 had significantly (p = 0.001) lower response to NAb production after than subjects having BMI < 30 after 180 days of immunization.  Factors associated with Nab titers after 30 and 180 days of the 2nd dosage of immunization. Table 3        www.nature.com/scientificreports/  Table 2.  ; p < 0.001). However, a notable increase of NAb titer (9.08 [4.10 to 14.06]; p < 0.001) was observed among COVID-19 positive subjects after 180 days of vaccination. We also performed another set of multivariable analysis keeping the COVID-19 positive and negative subjects in separate groups. We found similar significance in NAbs titer within the variables even if we keep the infected and uninfected subjects in separate groups (Supplementary Tables 1, 2).

Discussion
Virus-specific NAb have long been thought to be a key factor in viral clearance but this has been challenged as there is a continual propagation of increasingly dangerous variations of SARS-CoV-2 strains that can evade both vaccine-induced and convalescent immune responses 25 . The S glycoprotein's receptor-binding domain (RBD) and N-terminal domain (NTD) of SARS-CoV-2 detect and bind to the angiotensin-converting enzyme-2 (ACE-2) receptor, which is prerequisite for virus attachment and entry into host cells. The RBD, precisely the receptorbinding motif (RBM) region, also contains the main antigenic epitopes recognized by neutralizing antibodies (NAbs) 26,27 . Thus, any alteration in the S protein's structure can affect viral infection processes, providing the virus a selective advantage 28 . Therefore the Spike protein mutations are being extensively studied for their effects on disease transmission, pathophysiology, and vaccination efficacy as well 29 [34][35][36][37] . The efficacy of CoviShield (AZD1222) against Alpha variant was promising and well performing against Delta variant to some extent 38,39 . This neutralization effect of vaccine was further reduced for variants in other lineages and sub-lineages 40 . For example, the Omicron variants had the ability to evade immunity because of its higher affinity for human ACE2 than Delta variants, indicating a higher transmission 41 . Due to the changes, antibodies established against prior lineages of SARS-CoV-2 are less effective in variants such as Omicron and Delta 42 . The causes driving this "antigenic shift" are likely to become stronger when the majority of the population develops resistance to the virus through infection, vaccination, or both 43 . The SARS CoV-2 neutralizing antibody developed after immunization and retained in the blood for few months depending on several physiological factors 44 . Lower neutralization titer and reduced vaccine efficacy against distinct viral types have also been documented, in addition to the effect of declining neutralization titers with time 30,[45][46][47][48] . As a result, we emphasized on in vitro viral neutralization titer found in vaccinated individual in a cohort manner.
We revealed that after two dosage of immunization, the NAbs titer decreased over time in relation to age, Type 2 Diabetes and their lifestyles (including body-mass index, use of tobacco, and medication like corticosteroids for asthma and COPD patients). Our findings show that 30 days after receiving the 2nd dosage of AZD1222, adults' NAb titer against SARS-CoV-2 are enhanced differentially. Conversely, in adults, after 180 days, there was a considerable drop in NAb titer. This decreasing trend is most noticeable in people over the age of 50 and smokers who have type-2 diabetes and high BMI. In case of the diabetic patients, a low-grade metabolic inflammation is seen that is similar to chronic inflammation found in obese individuals. This inflammation may weaken macrophage activation and blunt the mechanism of cytokine production 49,50 . Besides the obese and type 2 diabetic people have more compromised B cell and T cell response 51,52 . Patients with T2DM also have a poor humoral immune response, making them more susceptible to re-infection 53 . After two dosage of the Pfizer-BioNTech BNT162b2 mRNA vaccine, the T2DM group showed significantly lower antibody titers than the non-diabetic group 44 . These partially explains the vaccine escape mechanism and rapid reduction of NAbs in people with type 2 diabetes and high BMI. Our findings show that NAb titer in patients were varied, and that the protective humoral immune response to SARS-CoV-2 may fade over time, similar to what has been observed in patients infected with other human coronaviruses such as HCoV-229E 54,55 . Patients infected with COVID-19 have a short-term humoral immune response that is very similar to that seen in patients infected with SARS-CoV and MERS-CoV 56,57 , who have a rapid drop in virus-specific antibody titers within 3-4 months.
We also found that the NAb titer decreases over time as well as depends on the age group and their lifestyles (including use of tobacco, and medication like corticosteroids for asthma and COPD patients). In case of smokers' , we found that 30 days after receiving the 2nd dosage of AZD1222, NAb titer against SARS-CoV-2 are enhanced differentially. There is a diminishing trend noticeable in smokers. Smoking has been shown to raise the expression of ACE2 58 , the SARS-CoV-2 virus's receptor for cellular entry, and to increase the risk of severe COVID-19 illness in young adults 59 . Although omicron infection is 40-70% less severe in young people than Delta infections irrespective of ACE2 expression 60 .
Neutralizing antibody has not been linked to a reduction in COVID-19 disease severity and lacks the response of cell mediated immunity 15,61-64 , as has been found in the case of Middle Eastern respiratory syndrome (MERS), which is caused by infection with the human coronavirus MERS-CoV 63 . However, in nonhuman primates, neutralizing antibody is linked to protective immunity against secondary (2°) infection with SARS-CoV-2 or SARS-CoV 15,[45][46][47][48] .Yet, the SARS CoV-2 neutralizing antibody is retained in the blood for several months. Lower neutralization titer and reduced vaccine efficacy against distinct viral types have also been documented, in addition to the effect of declining neutralization titers with time 30 www.nature.com/scientificreports/ Viral evolution is a continual process that, in the long term, might improve "viral fitness" and selective adaptation. As the variants of concern (VOCs) can be caused by new mutations in the S gene thus vaccine efficacy must be monitored on a continuous basis. If vaccines do not provide comprehensive protection against VOC variations, as with the H1N1 vaccine, periodic vaccine updates or reconstruction will be required. Novel vaccinations that generate NAbs against diverse variants by targeting highly conserved antigenic epitopes of the S protein are further options.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/